CRISPR Cas9 Lentiviral Guide RNA Custom Constructs and Libraries

The CRISPR-Cas9 system can be used for knocking out gene expression in vivo or in vitro by using a combination of an sgRNA (single guide RNA) along with Cas9 (dCas9) nuclease. Achieve permanent 100% knockout in your cell line by using Cellecta´s lentiviral-based CRISPR constructs. Expression of the sgRNA and Cas9 are stable and can be used in dividing or non-dividing cells or whole model organisms. Let our Partner Cellecta design and clone the sgRNA for you, or create an sgRNA library for your system. Cellecta´s HEAT sgRNA design makes it possible to substantially improve the quality of sgRNA libraries and screens: HEAT sgRNA Design!

Cellecta are offering a Single-Vector as well as a Two-Vector CRISPR-Cas9 System.

Single Vector CRISPR-Cas9 System: Stable Expression of Cas9 and sgRNA All-in-One-Vector
• Co-transduction not necessary since sgRNA and Cas9 nuclease are expressed in one vector
• Lentiviral vectors integrate into the host cell genome and are passed onto daughter cells
• Cells can be treated, grown for several passages, frozen and thawed - the lentiviral construct remains in the host cells
• Vector contains antibiotic selection to ensure stability

Two popular one-in-all sgRNA cloning vectors from Cellecta.
Please see link below for more choices.

More vectors, e.g. with EF1 or SV40 promoter, are also available.
Please inquire, when you do not find the promoter or marker combination of your choice.

Two Vector CRISPR-Cas9 System
The Two Vector CRISPR-Cas9 system is used to perform functional knockout screens in vivo or in vitro by expressing sgRNA (single guide RNA) and Cas9 nuclease from different lentiviral vectors.

The Cas9 Expression Vector is also available with blasticidin instead of hygromycin resistance.

Permanently Knock Out Your Targets with Custom Lentiviral sgRNA Constructs
• You provide the RefSeq number or Gene ID
• Any number of sgRNA constructs targeting the transcript are designed, synthesized, cloned into the vector of your choice and provided.
• Optionally, the lentiviral packaging of your custom constructs is also available as a service.

All Cellecta gRNA cloning vectors are available empty, either as supercoiled plasmid DNA, as linearized DNA / ready-to-clone your own gRNA, or packaged in lentiviral particles as negative controls in your experiment. See link below.
The Cas9 expression construct pR-CMV-Cas9-2A-Hygro for the Two-Vector System is available as plasmid DNA or as ready-to-transduce lentiviral particles.

Example Data: Knock Out of copGFP Gene in HEK293 Cells

Several sgRNAs targeting the copGFP gene were designed and individually transduced into HEK293 cells stably expressing copGFP. After 9 days, cells were imaged for GFP fluorescence. For each sgRNA tested, GFP expression was abolished in at least 70% of transduced cells.

After 9 or 14 days, cells were analyzed by FACS. GFP expression was abolished in at least 70% of the analyzed cells after day 9 and at least 90% after day 14.

Empty cloning vectors, CopGFP non-targeting controls, as well as several lethal sgRNA controls are available in all Cellecta vectors, either as plasmid DNA, or packaged in lentiviral particles. See the link Cellecta CRISPR Vectors and Controls below.

Custom Knock-out Cell Lines made with CRISPR Constructs
You tell us the RefSeq or other target gene identifier and the cell line-of-interest. Our partner Cellecta does the rest.

• Two clonal lines with knockout verified by genomic sequencing are returned
• Functional assays also possible

Our partner Cellecta has the expertise and capability to generate high quality complex heterogeneous libraries consisting of virtually any defined sequences. This capability combined with their proprietary vectors and lentiviral expertise provides Cellecta with the ideal platform to create excellent quality, highly representative pooled targeted or genome-wide lentiviral sgRNA libraries for CRISPR Cas9 knockout screens.
Cellecta provides custom-designed sgRNA libraries targeting any set of genes in which you are interested.

Vector Choice
Library Construction is offered in all Cellecta vectors, but it is highly recommended to apply Cellecta´s Dual Vector CRISPR System:
After introducing the Cas9 expression construct into a small population of cells, these can be selected strongly to express high levels of the nuclease so that, when the pooled sgRNA are introduced, gene knockout occurs more quickly and consistently.

Optionally, the lentiviral packaging of your custom library is also available as a service.